This invention relates to methods for binding a protein with an antibody reagent such as is accomplished in the performance of immunoassays. In particular, the invention concerns the binding of antibodies, fragments thereof and the like, to specific linear peptide epitopes in proteins and polypeptides.
There is a continuing need to improve the specificity of antibody binding to proteins, particularly proteins of analytical significance. The specific detection of particular proteins in biological samples such as blood is limited by the ability to obtain antibody reagents directed to unique binding sites or epitopes on the accessible portions of the proteins. There are situations where the most desirable epitope for specific detection of a particular protein is inaccessible or has only limited accessibility for binding to an antibody reagent.
An example is the determination of the glucosylated form of hemoglobin known as Hb Alc in blood samples of diabetic patients. Hemoglobin is a protein tetramer made up of four chains (subunits) of amino acids, each of about 143 units and having a total molecular weight of about 64,000. At one end of the molecule (the NH.sub.2 -terminus of the beta-subunit) there is a valine unit which can react with glucose. The glucosylation of hemoglobin occurs by a non-enzymatic reaction involving glucose and the alpha-amino group of valine. Following a Schiff base formation between the reactants, the glucose undergoes an Amadori rearrangement forming 1-deoxyfructo-valine. This complex is covalent and essentially irreversible. The glucosylation reaction is governed by the concentration of the reactants, e.g., hemoglobin and glucose. In a normal (non-diabetic) individual approximately 3% of the total hemoglobin is glucosylated. Hemoglobin tetramers with a 1-deoxyfructo-valine on the N-terminus of a beta-chain are identified as being glucosylated or A.sub.1c hemoglobin.
Glucose levels in diabetics are sufficiently high to increase the rate of glucosylation in direct dependence upon the glucose level in the blood, which reflects the severity of the diabetic condition. With hemoglobin, the A.sub.1c level is raised to about 5 to 12%. Since the circulating life span of hemoglobin is about 120 days, a glucosylated hemoglobin measurement will give a value which reflects an average glucose level for that period. Notably a meal high in glucose will not be reflected in a high glucosylated hemoglobin or serum albumin level. Thus, measurement of the glucosylated hemoglobin content gives a truer picture of the average circulating glucose levels and thus a truer picture of the long term condition of the patient.
U.S. Pat. No. 4,247,533 discloses an analytical technique wherein antibodies to Hb A.sub.1c were reportedly raised in a special sheep by injection of Hb A.sub.1c and absorbed with nonglucosylated hemoglobin to provide polyclonal antibodies which distinguished between Hb A.sub.1c and nonglucosylated Hb. Such antibodies then form the basis for a test to determine the proportion of glucosylated hemoglobin in a sample. The test, however, requires an appropriately immunized sheep and antibody absorptions to attain the proper specificity. It is, therefore, costly and difficult to produce specific polyclonal antibodies. The antibody preparations produced by this absorption approach are reported to be of low titer and affinity. The reproducibility of this approach is also open to question since there are no recent reports describing its use for the analysis of clinical samples of human hemoglobin.
Another attempt to obtain antibodies specific for Hb A.sub.1c id found in U.S. Pat. No. 4,478,744. These workers substituted a synthetic peptide immunogen for the normal hemoglobin molecule as the immunizing agent. This material was injected into an animal which normally does not have Hb A.sub.1c in its bloodstream, e.g., sheep. The synthetic peptide immunogen comprised a glucosylated peptide residue having an amino acid sequence corresponding to between the first 4 to 10 amino acids in the N-terminal hemoglobin sequence. Subsequent investigations, reported hereinbelow, have found that the sheep polyclonal antiserum raised against the synthetic peptide immunogen has no detectable specificity for the glucosylated form, Hb A.sub.1c.
Therefore, there is an unsatisfied need to develop an approach to designing antibody reagents and binding conditions that permit the specific binding of antibody reagents to proteins of interest. This is particularly apparent from the inability of prior workers to devise immunoassays for the determination of particular proteins such as glycosylated proteins, e.g., Hb A.sub.1c.
______________________________________ Definitions Amino Acid Abbreviation ______________________________________ Arginine Arg Asparatic Acid Asp Glutamic Acid Glu Lysine Lys Serine Ser Asparagine Asn Glutamine Gln Glycine Gly Proline Pro Threonine Thr Alanine Ala Histidine His Cysteine Cys Methionine Met Valine Val Isoleucine Ile Leucine Leu Tyrosine Tyr Phenylalanine Phe Tryptophan Trp ______________________________________